Gregorio García-Herdugo

Growth kinetics of the Golgi apparatus during the cell cycle in onion root meristems

In onion root meristems, the number of dictyosomes per cell shows a kinetics of growth strongly related to the cell cycle. During the interphase of steady-state proliferative cells, the volume density and numerical density of the Golgi apparatus decrease to reach minimum values in late-interphase cells, characterized by their greatest length. This pattern is also found in the total volume occupied by Golgi apparatus. Once in mitosis, the above-mentioned parameters begin to increase reaching maximum mean values in telophase. After the experimental uncoupling of chromosome and growth cycles by presynchronization with hydroxyurea, we found a similar behaviour pattern in the Golgi apparatus: decreasing values during interphase and a triggering of Golgi-apparatus growth in prophase independently of the bigger cell sizes reached in mitosis as an effect of pretreatment with hydroxyurea. These results indicate a cyclic kinetics of this subcellular component in higher-plant meristems, coupled with early mitotic events.

Characterization and immunolocalization of a nucleolar antigen with anti-NOR serum in HeLa cells.

We have used a serum from a patient with rheumatoid arthritis and found it to immunoblot with a 92- to 88-kDa protein doublet with an isoelectric point of around 7.5 after mono- and two-dimensional electrophoresis in whole HeLa cells. By means of immunofluorescence and immunoelectron microscopy we have found it to specifically react with the nucleolar fibrillar component. After quantitative analysis under the electron microscope, we have demonstrated a similar labeling both in the fibrillar centers and the dense fibrillar component, using two different gold-coupled markers. When transcription was inhibited under physiological conditions (mitosis) or after AMD treatment the antigen remained, as shown by immunoblotting and immunolabeling with anti-NOR serum. These biochemical characteristics, which coincide with those of the ribosomal transcription human upstream binding factor, together with the immunolocalization with anti-NOR serum, allow us to discuss the possible role of these antigens in rDNA transcription.

Ag-NOR proteins and rDNA transcriptional activity in plant cells.

In this work we used Allium cepa root meristem cells in actively growing conditions and under treatment with the protein synthesis inhibitors cycloheximide (CHM) and puromycin. Morphological and quantitative results indicate that these drugs induce dramatic alterations in nucleolar structure reflected by a decrease of nucleolar size, much more evident under treatment with CHM, and by segregation of its main components. Quantitative analysis shows a decrease in NOR-silver staining after treatment with CHM, whereas in cells treated with puromycin NOR-silver staining remains constant. Our results reveal a decrease in the Ag-NOR proteins under conditions of diminished cell activity, suggesting a direct relationship between the quantity of Ag-NOR proteins and transcriptional activity. Using two-dimensional gel electrophoresis and NOR-silver staining in gels, we have characterized some proteins corresponding to molecular weights of 28 and 31 KD and pI of approximately 5.2. After treatment with CHM, reactivity of these proteins against NOR-silver staining is diminished. By means of a morphological study, analysis of NOR-silver staining, and of anti-DNA and RNAse-gold labeling, we have tried an approach to the nucleolar organization in plant cells. Our results suggest that the fibrillar component shows a reticular distribution where fibrillar centers, as described in animal cells, are not distinguished.

DNA STRAND‐BREAKS INDUCED BY THE TOPOISOMERASE I INHIBITOR CAMPTOTHECIN IN UNSTIMULATED HUMAN WHITE BLOOD CELLS

Camptothecin (CPT) and actinomicyn‐induced strand‐breaks, repair and apoptosis in unstimulated human blood cells were studied using the DNA comet assay, and electrophoresis of low molecular weight DNA extracts. On the one hand, incubation of G0 leukocytes for 1h with CPT induced DNA strand‐breaks that were observed using the single cell gel electrophoresis technique. On the other hand, internucleosomal DNA fragments were not observed, suggesting that apoptosis had not occurred. DNA‐strand‐breaks caused by CPT were repaired 24h after treatment; the migration of DNA fragments was assessed by a reduction in the number of comets. These data strongly suggest that the unexpected clastogenic effect of this topoisomerase I inhibitor is not due to the collision of the cleavage complex with the replication fork, since replication does not occur in G0. In our opinion, this effect could be due instead to the topoisomerase I enzyme being able to bind DNA in the absence of replication, probably in a way that is not strictly related to the progression of the cell cycle. Thus, CPT does not provoke apoptosis in quiescent leukocytes.

Nucleolar component behaviour in plant cells under different physiological conditions. A morphological, cytochemical and quantitative study

Different nucleolar components of Allium cepa L. root meristem cells under physiological conditions of proliferation and quiescence were quantitatively analyzed after the application of morphometric and cytochemical techniques.

Inhibition of nucleolar protein nucleolin by electroporation with anti‐nucleolin antibodies results in an increase of the nucleolar size

Electroporation of exponentially growing human larynx epidermoid carcinoma cells (HEp‐2) with a serum against nucleolin, one of the most abundant non‐histone nuclear proteins, has shown, 24 h after electroporation, a significant increase in the size of the nucleolus of these cells compared with normal HEp‐2 cells (non‐electroporated) and electroporated HEp‐2 cells in the absence of antinucleolin serum (P < 0.01). Image analysis evaluation of the different nucleolar components proved a major contribution of the dense fibrillar component to the total nucleolar size in cells electroporated with anti‐nucleolin antibodies, more than that corresponding to the dense fibrillar component in cells from any of the control groups (P < 0.01), indicating that the reported increase in nucleolar size was due to a marked enlargement of the dense fibrillar regions. These results, in agreement with previous biochemical and molecular biology studies, suggest a pivotal role for nucleolin in pre‐rRNA processing and constitute morphological evidence supporting this role. Following nucleolin inhibition, impaired pre‐rRNA processing might result in an accumulation of this molecular species in the dense fibrillar component of the nucleolus, where pre‐rRNA is first present.

An electron microscopic study of stellate cells and cavities in the pars distalis of frog pituitary

Stellate cells in the pars distalis of adult Rana ridibunda were observed electron microscopically under normal and experimental conditions (TRH injection). The stellate cells have lengthy processes extending into the intercellular spaces between the secretory cells and scanty cytoplasm surrounding the nucleus. Occasional desmosomes link stellate cells to adjacent secretory cells. In the pars distalis of animals injected with thyrotropic-releasing hormone (TRH), the stellate cells form large cavities (2-6 mum) filled with heterogeneous material. Their cytoplasm contains well-developed Golgi complexes and some lysosomes; these are the principal morphological alterations as compared to those observed in control animals. It is suggested that stellate cells could play an active role in addition to providing a structural framework for the pars distalis.

In vitro expression and redistribution of nucleolar proteins following the treatment with cis-dichloro-1,2-propylenediamine-N,N,N',N'-tetraacetato ruthenium (III) (RAP).

In this study, we used a newly synthesized antitumor complex [RuLCl2]H.4H2O (RAP), having the same antitumor effects as cisplatin but showing lower cytotoxicity. We found that RAP-DNA adducts induce a high expression of proteins with high molecular weight and a low expression of proteins with low molecular weight. We choose two proteins: the upstream binding factor (UBF), an RNA polymerase I-specific transcription factor that recognizes the ribosomal RNA gene promoter and initiates transcription; and fibrillarin, which is involved in many posttranscriptional processes including pre-rRNA processing, pre-rRNA methylation, and ribosome assembly. Our results showed that UBF was present in high quantities in TG cell extracts treated with RAP with a major abundance of UBF1 more than UBF2, which was explained by a high affinity of UBF1 for DNA modified by RAP than UBF2; while fibrillarin was present in low quantities in protein extracts treated with RAP. Also, following treatment with RAP, there was a similar redistribution of UBF along the nucleus of TG cells as in the controls but with the presence of higher quantities of this factor in the nucleoplasm, which could be explained by an increase of the UBF affinity for the no nucleolar chromatin as a consequence of the modifications induced by RAP. Fibrillarin was found in low quantities in the fibrillar centers and in the nucleoplasm after treatment with RAP.

Changes of dictyosome ultrastructure during the cell cycle in onion root meristematic cells. A morphometric and stereological study

SummaryDictyosome ultrastructure changes during the cell cycle in onion root meristematic cells. Changes were mainly related to cisternae and intercisternal spaces morphology. Taking each dictyosome to be composed of three different regions (CIS, medial, and TRANS), several quantitative changes were detected in some of the compartments. Many of the planimetric parameters evaluated showed higher values in medical cisternae, while CIS and TRANS remained nearly constant. We have also found an increased activity of dictyosomes, as indicated by increase in the volume fraction of vesicular attached structures. This reaches maximum values at ana-telophase in coincidence with the onset and progression of cytokinesis.